Studies On Fermentation Conditions,Properties And In Vitro Activities Of Extracellular Yellow Pigment By Lachnum DP5

In this thesis,the fermentation conditions,purification,structure,physicochemical properties and in vitro activities of Lachnum DP5 yellow pigment were studied.Lachnum DP5 is a kind of important fungal resources which distributed all over the world,can produce extracellular yellow pigment under the condition of deep culture.The effects of carbon source,nitrogen source,inorganic salts,initial p H,growth time,temperature,speed and liquid volume on the pigment yield were studied by single factor experiments.According to the results of single factor experiment,the compositions of culture medium and fermentation conditions were studied by orthogonal experiments.Using 1% glycerol,0.3% yeast powder and 0.03% Mg Cl2 as the medium,the color value of yellow pigment increased by 1.79 times from 14.65 U/m L to 26.23 U/m L,at 75 m L,28 ℃,140 r/min,for 8 days.Extracellular yellow pigment was purified by organic solvent extraction and column chromatography.The effects of organic solvent,ratio of material to liquid and extraction times on the extraction efficiency of yellow pigment were studied,and the effects of column chromatography packing and elution method on the purification of yellow pigment were studied.The purity of yellow pigment was identified by thin layer chromatography（TLC）and high performance liquid chromatography（HPLC）.Crude yellow pigment LPY was obtained by extracting fermentation broth with petroleum ether（1:1,v/v）,twice.LPY-3 was separated by silica gel G column with petroleum ether and ethyl acetate elution system,and LPY-3d was separated by Rf C18 column with water and methanol elution system.LPY-3d was determined as a single component by HPLC and TLC with a purity of 99.01%.The structure of yellow pigment LPY-3d was analyzed by means of HPLC,TLC,UV-Vis,FT-IR,ESI-MS and NMR as follows:The stability of yellow pigment was studied by the effects of p H,temperature,light,metal ions,oxidants,reductants and food additives on the absorbance of yellow pigment.The solubility of yellow pigment was studied by using different solvents,and the thermal denaturation of yellow pigment was studied by DSC.The results showed LPY-3d will fade rapidly at p H < 5:00 and had good stability at p H > 5,and could increase color in alkaline environment,which stored in outdoor light,indoor light and temperature above 70 ℃,the fading phenomenon was obvious.LPY-3d was stable in K+,Zn²⁺,Mg²⁺ and Ca²⁺,and could increase color.It was easy to be broken in Al3+,Cu²⁺,Fe²⁺ and Fe3+,it had poor stability to VC and H2O2,and better stability to Na NO2 and Na2SO3.The stability of LPY-3d was better in glucose,soluble starch,Na Cl,sucrose which was poor in citric acid and potassium sorbate.The results of DSC showed that it began to melt at 86℃ and decomposed at 108 ℃ .Antioxidant activities of yellow pigment were studied by salicylic acid assay,DPPH assay,reductive capacity assay and soybean oil auto-oxidation assay.The antimicrobial activity of yellow pigment was studied by paper diffusion assay.The IC50 values of LPY-3d on the ·OH,DPPH,NO₂^-scavenging ability and reducing power were 220.091 μg/m L,23.961 μg/m L,1.152 μg/m L and 74.01 μg/m L,respectively.H2O2（300 μM）induced damage in Hep G2 cells for 2 h,and the cell survival rate was 49.50% by MTT method.The cell viabilities of LPY-3d treated groups（10,20 and 50 μg/m L）were 64.89%,66.57% and 81.71%,respectively.In 50 μg/m L concentration of LPY-3d,the SOD and CAT activities of Hep G2 cells were detected were 39.98 ± 3.02 U/mg,9.94 ± 1.36 U/mg,which were higher than those in the model group.MDA level was 2.12 ± 0.06 mg /mg,lower than the model group.LPY-3d had inhibitory effect on the growth of Escherichia coli,Staphylococcus aureus and Bacillus subtilis.